Hargrove, Amanda EMcFadden, Emily Josephine2021-01-122023-01-112020https://hdl.handle.net/10161/22181<p>The noncoding RNA (ncRNA) revolution has revealed myriad RNA species that play critical roles at all stages of life, including embryogenesis and disease progression. For example, three long ncRNA (lncRNA), Hox Transcript Antisense Intergenic RNA (HOTAIR; ~2.5 kb), Metastasis Associated Lung Adenocarcinoma Transcript-1 (MALAT1; ~6.7 kb) and Second Chromosome Locus Associated with Prostate-1 (SChLAP1; ~1.5 kb), are basally expressed in normal prostate tissue but are dysregulated in prostate cancer. HOTAIR scaffolds the PRC2 and LSD1 protein complexes to selectively methylate and demethylate, respectively, histone proteins, thereby regulating downstream gene expression. MALAT1 acts in trans at nuclear speckles during mRNA post-transcriptional processing while SChLAP1 acts in cis to influence oncogenic gene expression. Initial work on HOTAIR developed technical skills that were then applied to the lncRNAs MALAT and SChLAP1 to learn more about their role in prostate cancer. There is compelling evidence that the 3′–end of MALAT1 is a triple helix structure that acts as a molecular knot, driving transcript accumulation in cancer cells and furthering their metastatic potential, but we currently lack any biophysical data detailing the relationship between SChLAP1 structure and function. In general, the relationship among lncRNA structure, dynamics, and function is not well understood; for example, even with high-resolution structures of the MALAT1 triple helix, questions remain regarding the role of intrinsic dynamics in transcript stability or protein binding. As lncRNA represent an underexplored therapeutic avenue, this work aims to investigate the role of lncRNA structure and dynamics in driving prostate cancer metastasis. This work uses biophysical and biochemical methods including chemical probing, NMR, SAXS, and native gels to study the two lncRNA MALAT1 and SChLAP1 and learn more about their respective structure-function relationships. From this work, we have found a discrete structure within the lncRNA SChLAP1 that is highly structured and implicated in driving metastasis via protein recognition. Additionally, our preliminary studies regarding MALAT1 support the presence of non-triplex states that require further characterization. Overall, this work supports a deeper understanding of lncRNA structure as it relates to their function in cancer and provides examples for the biophysical analysis of large and/or structurally complex RNA. </p>BiochemistrylncRNAProstate cancerStructural biologyDissertation